State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat‑sen University, Guangzhou, Guangdong 510000, P.R. China.
Mol Med Rep. 2017 Nov;16(5):6974-6980. doi: 10.3892/mmr.2017.7475. Epub 2017 Sep 12.
The present study aimed to identify whether FK506 suppresses hypoxia‑induced inflammation and protects tight junction function via the calcineurin‑nuclear factor of activated T‑cells 1 (CaN‑NFATc1) signaling pathway in mouse retinal microvascular endothelial cells (mRMECs). The mRMECs were treated with FK506 at different concentrations following the induction of hypoxia. Trans‑epithelial electrical resistance (TEER) and cell permeability were examined to measure the integrity of the tight junctions. The concentrations of inflammatory cytokines were measured using reverse transcription‑quantitative polymerase chain reaction analysis and enzyme‑linked immunosorbent assays. The protein expression levels of zonula occludens‑1 (ZO‑1) and nuclear factor of activated T‑cell 1 (NFATc1) were identified using immunofluorescent microscopy and western blot analysis. The TEER value was decreased following hypoxia, but increased following treatment with FK506 (1 and 10 µM) for 24 and 48 h. The protein expression of ZO‑1 was also increased following FK506 treatment for 24 h at 1 and 10 µM. By contrast, following treatment with FK506 (1 and 10 µM) for 24 and 48 h, the elevated cell permeability in the hypoxia group was significantly downregulated. Similarly, the concentrations of inflammatory cytokines, including cyclooxygenase‑2, inducible nitric oxide synthase, monocyte chemoattractant protein‑1, interleukin‑6, intercellular adhesion molecule‑1 and vascular cell adhesion molecule‑1, were downregulated following treatment with FK506 for 24 h at 1 and 10 µM. Following treatment with FK506, the level of total NFATc1 was downregulated and the level of phosphorylated NFATc1 was upregulated. Taken together, FK506 suppressed injury to the tight junctions and downregulated the expression of inflammatory cytokines in hypoxia‑induced mRMECs via the CaN‑NFATc1 signaling pathway. This suggests a potentially effective therapy for hypoxia‑induced retinal microangiopathy.
本研究旨在探讨 FK506 是否通过钙调神经磷酸酶-活化 T 细胞核因子 1（CaN-NFATc1）信号通路抑制缺氧诱导的炎症反应，保护紧密连接功能，从而减轻小鼠视网膜微血管内皮细胞（mRMECs）的损伤。 mRMECs 在缺氧诱导后用不同浓度的 FK506 处理。通过跨上皮电阻（TEER）和细胞通透性测定来测量紧密连接的完整性。采用逆转录-定量聚合酶链反应分析和酶联免疫吸附试验测定炎症细胞因子的浓度。采用免疫荧光显微镜和 Western blot 分析检测闭锁蛋白-1（ZO-1）和活化 T 细胞核因子 1（NFATc1）的蛋白表达水平。缺氧后 TEER 值降低，但用 FK506（1 和 10 μM）处理 24 和 48 小时后 TEER 值增加。用 FK506（1 和 10 μM）处理 24 小时后，ZO-1 的蛋白表达也增加。相反，用 FK506（1 和 10 μM）处理 24 和 48 小时后，缺氧组细胞通透性的升高显著下调。同样，用 FK506 处理 24 小时后，炎症细胞因子（包括环氧化酶-2、诱导型一氧化氮合酶、单核细胞趋化蛋白-1、白细胞介素-6、细胞间黏附分子-1 和血管细胞黏附分子-1）的浓度也降低。用 FK506 处理后，总 NFATc1 水平下调，磷酸化 NFATc1 水平上调。综上所述，FK506 通过 CaN-NFATc1 信号通路抑制缺氧诱导的 mRMECs 中紧密连接损伤和炎症因子表达，为缺氧诱导的视网膜微血管病变提供了一种潜在有效的治疗方法。
